Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, S.
Right arrow Articles by Roman, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, S.
Right arrow Articles by Roman, J.
Clinical Cancer Research Vol. 10, 1911-1919, March 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Up-regulation of p21 Gene Expression by Peroxisome Proliferator-Activated Receptor {gamma} in Human Lung Carcinoma Cells

Shouwei Han1, Neil Sidell2, Paul B. Fisher3 and Jesse Roman1,4

1 Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine and 2 Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia; 3 Departments of Pathology, Neurosurgery, and Urology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, New York; and 4 Atlanta Veterans Affairs Medical Center, Atlanta, Georgia

ABSTRACT

Purpose: The peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the regulation of cell growth and differentiation although the exact mechanism(s) of this activity has not been elucidated. In this study, we explored the role of PPAR{gamma} signaling on the control of gene expression of the cycle-dependent kinase inhibitor p21 in human lung carcinoma cells.

Experimental Design: Using several human lung carcinoma cell lines (small and non-small carcinoma cells), we assayed for cell growth inhibition and apoptosis induction. We also assayed for p21 mRNA and protein expression by reverse transcription-PCR, real-time reverse transcription-PCR, and Western blot analysis. Nuclear protein binding activities to three response elements located in the p21 promoter [nuclear factor (NF)-{kappa}B, Sp1, and NF-interleukin 6 (IL6) CAAT/enhancer binding protein (C/EBP)] were measured by gel mobility shift assays. We used transient transfection assays with p21 promoter reporter gene constructs to determine the transcriptional regulation by PPAR{gamma} ligands. Finally, by using p21 antisense oligonucleotides, we tested the link between PPAR{gamma} activation and p21 signaling in cell growth inhibition assays and by Western blot analysis.

Results: We showed that the PPAR{gamma} ligands PGJ2 and ciglitazone inhibit the growth and induce the apoptosis of several human lung carcinoma cell lines, whereas the PPAR{alpha} agonist WY14643 has little effect. Treatment of lung carcinoma cells with the PPAR{gamma} ligands PGJ2, ciglitazone, troglizaone, and GW1929 elevated p21 mRNA and protein levels and reduced cyclin D1 mRNA levels. These results were supported by transient transfection assays, which indicated that PPAR{gamma} ligands increased p21 gene promoter activity in human lung carcinoma cells. In addition, p21 antisense oligonucleotides inhibited PPAR{gamma} ligand-induced p21 protein expression and significantly blocked lung carcinoma cell growth inhibition induced by PPAR{gamma} ligands. Finally, electrophoresis mobility shift experiments demonstrated that PPAR{gamma} ligands increased the nuclear binding activities of Sp1 and NF-IL6 (C/EBP), two transcription factors with regulatory elements in the promoter region of the p21 gene.

Conclusion: PPAR{gamma} ligands inhibit human lung carcinoma cell growth and induce apoptosis by stimulating the cyclin-dependent kinase inhibitor p21 and by reducing cyclin D1 gene expression. The induction of p21 gene expression by PPAR{gamma} ligands may be mediated through increased Sp1- and NF-IL6 (C/EBP)-dependent transcriptional activation. These observations unveil a mechanism for p21 gene regulation in lung carcinoma that represents a potential target for therapy.

Introduction

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (1 , 2) . A family of negative cell cycle regulators, cyclin-dependent kinase inhibitors, modulates these events, especially those controlling the transition of G1 to S-phase (2) . The latter includes two families, the CIP/KIP family and the INK4 family. p21 is a member of the CIP/KIP family (2 , 3) and plays a crucial role in growth arrest induced in transformed and normal cells by a variety of mechanisms (4) . p21 was identified and cloned in a number of cellular systems including p53-dependent growth arrest and DNA-damage induction (5, 6, 7) , p53-independent melanoma (mda-6; 8 , 9 ), leukemia cell differentiation (10 , 11) , and during cellular senescence (sdi-1) in human skin fibroblasts (12) . p21 is poorly expressed in quiescent cells; it is rapidly induced when cells are stimulated with mitogens, and its expression then declines as cells reach mid-late G1 phase (2 , 13) . Although many studies have implicated p21 in cell cycle regulation, especially in G1 arrest, the clinical significance of p21 in human lung carcinoma is still controversial (14) . However, in patients with non-small cell lung carcinoma, p21 expression was shown to be an independent prognostic factor (15 , 16) , and it may represent an important clinical marker of outcome in non-small cell lung carcinoma. We believe that p21 is a potent modulator of lung carcinoma cell growth as well as an important marker in the prognosis of this disease. It is for this reason that we have focused our attention on this cell cycle gene and how its expression is affected by peroxisome proliferator-activated receptor (PPAR) ligands.

The PPAR{gamma} is a member of the nuclear receptor superfamily of ligand-dependent transcriptional factors (17) . PPAR{gamma} plays an important role in the differentiation of adipocytes and monocytes/macrophages, as well as in cell proliferation, apoptosis, and carcinoma cell arrest (17) . In humans, PPAR{gamma} expression is detectable in normal tissues including brain, breast, prostate, colon, lung, ovary, and placenta (18, 19, 20, 21) . Recent studies revealed that PPAR{gamma} ligands can cause growth inhibition by inducing the differentiation or by promoting the apoptosis of several leukemia cells and solid tumors including breast, prostatic, colorectal, gastric, and lung carcinoma (20 , 22, 23, 24, 25) . Of note, modulation of p21 expression by PPAR{gamma} ligands has been shown in several cell types (26, 27, 28) . However, the mechanisms by which PPAR{gamma} activation regulates G1 phase arrest and p21 gene expression remain to be elucidated.

Herein, we show that PPAR{gamma} ligands inhibit human lung carcinoma cell growth and induce apoptosis by stimulating the expression of the cyclin-dependent kinase inhibitor p21 and by reducing the expression of cyclin D1. These results suggest that the induction of p21 by PPAR{gamma} ligands is attributable to increased transcriptional activation of this gene mediated by an increase in Sp1 and nuclear factor-interleukin 6 (NF-IL6) CAAT/enhancer binding protein (C/EBP) binding activity.

Materials and Methods

Culture and Chemicals.
The human small cell lung carcinoma cell lines (H345, H2081) and non-small cell lung carcinoma cell lines (H1838, H2106) were obtained from the American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 supplemented with 10% heat-inactivated FBS, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg of amphotericin (complete medium) as described previously (29) , The PGJ2 and ciglitazone (Cig) were purchased from Alexis Biochemical (San Diego, CA). The PPAR{gamma} antagonist GW9662 (30) was obtained from Cayman Chemical Co. (Ann Arbor, MI). The dual-luciferase report assay kit, the gel shift assay system and 5' DNA terminus-labeling system were obtained from Promega (Madison, WI). The p21 and the cyclin D1 polyclonal antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). The [{gamma}-32P]ATP was purchased from Perkin-Elmer Life Sciences, Inc. (Boston, MA). TACs Apoptotic DNA laddering kit was obtained from R&D Systems (Minneapolis, MN). The GW1929 and WY14643 were purchased from Calbiochem (San Diego CA). The PPAR{gamma} polyclonal antibody and the troglitazone were obtained from Biomol Research Laboratories, Inc (Plymouth Meeting, PA). The LightCycler-FastStart DNA Master SYBR Green 1 kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). All other chemicals were purchased from Sigma Chemicals, Inc. (St. Louis, MO) unless otherwise indicated.

Cell Growth Inhibition Assay.
H2106 human lung carcinoma cells (105 cells/well) were seeded in 6-well culture plates and incubated with various concentrations of PGJ2, Cig, or WY14643 for up to 4 days. The cells were subsequently harvested by trypsinization using trypsin/EDTA and counted under the microscope after trypan-blue staining.

Reverse Transcriptase (RT)-PCR.
Total RNA was prepared from human lung carcinoma cells with TRIzol Reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s instructions. To amplify 474 bp PPAR{gamma}, 354 bp p21, 323 bp cyclin D1, and 200 bp glyceraldehyde-3-phosphate dehydrogenase cDNA fragments, the sequences of PCR primers (Sigma Genosys, Woodlands, TX) were as follows: for PPAR{gamma} sense (5'-CTCTCCGTAATGGAAGACC-3'), antisense (5'-GCATTATGAGACATCCCCAC-3'); for p21 sense (5'-GCGATGGAACTTCGACTTTGT-3'), antisense (5'-GGGCTTCCTCTTGGAGAAGAT-3'); for cyclin D1 sense (5'-GGCAACGGAGGTCTGCG-3'), antisense (5'-GTCGGTGGTAGATGCACAGCTT-3'); and for glyceraldehyde-3-phosphate dehydrogenase sense (5'-CCATGGAGAAGGCTGGGG-3'), antisense (5'-CAAAGTTGTCATGGATGACC-3') according to published data (31, 32, 33) . RT-PCR was carried out as described previously (31) . The samples were first denatured at 95°C for 30 s, followed by 32 PCR cycles, each with temperature variations as follows: 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The last cycle was followed by an additional extension incubation of 7 min at 72°C. Analysis of amplicons was accomplished on 1% agrose gel containing 0.2 µg/µl ethidium bromide and visualized under UV transilluminator. The densitometric analysis of PCR products was performed by computer software (Bio-Rad Quantity One), GS-800 Imaging Densitometer (Bio-Rad, Hercules, CA), and standardized to the glyceraldehyde-3-phosphate dehydrogenase product.

Real-Time RT-PCR.
This procedure, which was based on the time point during cycling when amplification of the PCR product was first detected, rather than on the amount of PCR product accumulated after a fixed number of cycles, was described previously (34) . Final results were expressed as n-fold differences in p21, or cyclin D1 gene expression relative to the glyceraldehyde-3-phosphate dehydrogenase gene. All PCR reactions using LightCycler-FastStart DNA Master SYBR Green I kit were performed in the Cepheid Smart-Cycler real-time PCR cycler (Sunnyvale, CA; Ref. 34 ). Experiments were performed in triplicate for each data point.

Detection of DNA Fragmentation.
Genomic DNA was isolated from human lung carcinoma cells using TACS Apoptotic DNA Laddering Kits (R&D Systems) to evaluate apoptotic cell death. Procedures for DNA isolation and detection followed the protocol supplied by the product supplier as we described before (29) . After running in a 1.5% TreviGel 500 gel [% (w/v)] in 1x Tris-acetate-EDTA, DNA was visualized with ethidium bromide staining under a UV transilluminator and photographed.

Oligodeoxynucleotide Transfections.
Phosphorothioate p21 antisense and random sequence control oligodeoxynucleotides (ODN) designed around the translation start site were synthesized by Sigma Genoys according to published data (35) . The p21 antisense sequence was 5'-GAC ATCACCAGGATCGGACAT-3'. The random sequence control was 5'-TGGATC CGACATGTCAGA-3'. For the transfection procedure, cells were grown to 60% confluence, and the 2 µg/µl concentration of oligodeoxynucleotide was mixed with 3 µl of FuGENE 6 transfection reagent (Roche per well of serum-free medium and was added to the cells for 24 h at 37°C. The media was changed into regular culture medium in the morning, and the cells were treated with PPAR{gamma} ligands for 24 and 48 h for Western Blot and cell growth assay, respectively.

Western Blot Analysis.
The procedure was performed as described previously (36) . Protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein (50 µg) from whole cell lysates were solubilized in 2x SDS-sample buffer, separated on SDS polyacrylamide gels. The separated proteins were transferred onto nitrocellulose and blocked with Blotto {1x Tris-buffered saline [10 mM Tris HCl (pH 8.0), 150 mM NaCl]} with 5% nonfat dry milk and 0.1% Tween 20 for overnight at 4°C. Blots were incubated with antibodies raised against rabbit p21 (1:500), cyclin D1, or PPAR{gamma} (1:1000 dilution) for 2 h at room temperature, then incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution; Sigma) for 1 h at room temperature. The blots were washed, transferred to freshly made enhanced chemiluminescence solution (Amersham, Arlington, IL) and exposed to X-ray film. Protein bands were quantified by densitometric scanning using a Bio-Rad GS-800-calibrated densitometer. In controls, the p21, cyclin D1, and PPAR{gamma} antibodies were omitted or replaced by serum IgG.

Plasmids.
The 2.3-kb fragment of the p21 promoter construct ligated to flyfire luciferase reporter gene has been reported previously (37) . Synthetic Renilla luciferase report vector (phRL-SV40) was obtained from Promega.

Transient Transfection Assays.
Human lung carcinoma cells were seeded at a density of 1 x 105 cells/well in 6-well dishes and grown to 60% confluence. For each well, the plasmid DNA containing wild-type p21 promoter construct, and 0.2 µg of the internal control plasmid phRL-SV40 (Renilla luciferase gene) were cotransfected into the cells using 6 µl of FuGENE 6 lipofection reagent as described in our earlier work (38) . After 24 h of incubation, cells were treated with PPAR{gamma} ligands and PPAR{alpha} ligand for an additional 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the dual-luciferase reporter kit according to recommendations by the manufacturer. The assays for firefly luciferase activity and Renilla luciferase activity were performed sequentially using two reaction tubes in a luminometer with two injectors (Thermo Labsystems, Helsinki, Finland). Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity in the same sample.

Electrophoretic Mobility Shift Assay.
Nuclear protein extracts were prepared for electrophoretic mobility shift assay as described earlier (39) . The protein content of the nuclear extract was determined using the Bradford protein assay kit (Sigma). Electrophoretic mobility shift assay experiments were performed as described previously (29) . The probes of double-stranded oligonucleotides for Sp1 and NF-IL6 (C/EBP) that were synthesized by Sigma-Genosys based on human p21 promoter sequence (37 , 40) were, as follows: wild-type Sp1 (5'-GGGGGTCCCGCCTCCTTGA-3'); mutant Sp1 (5'-GGGGGTC-gacCCTCCTTG-3'). Wild-type NF-IL6 (C/EBP; 5'-GTACTTAAGAAATATTGAAT-3'); C/EBP mutant (5'-GTACaaAAGAAATATTGAAT-3'). The underlined bases indicate mutations. The complimentary oligonucleotides were annealed and purified following the manufacturer’s instructions. The Sp1 and NF-IL6 (C/EBP) oligonucleotides were end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase as recommended by the manufacturer. Ten µg nuclear proteins from control and treated cells were incubated with 32P-labeled oligonucleotide probe under binding conditions (Promega) for 20 min at room temperature in a final volume of 20 µl. For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotides was added in reaction buffer contained nuclear protein for 10 min before adding probe. The same amount of mutated oligonucleotide probe or 100-fold excess of mutated oligonucleotides was used as another control. After binding, protein-DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel using 1x Tris-glycine buffer. Each gel was then dried and subjected to autoradiography at -80°C.

Statistical Analysis.
All experiments were repeated a minimum of three times. All data collected from electrophoresis gel mobility shift assays, luciferase activity assays, RT-PCR, or real-time RT-PCR and Western blot were expressed as means ± SD. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student’s t test (two-tailed) comparison between two groups of data sets. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05, see Fig. legends).

Results

PPAR{gamma} Gene Expression in Human Lung Carcinoma Cells.
To confirm that PPAR{gamma} is expressed in the lung carcinoma cell lines studied, we examined its expression by RT-PCR and Western blot analyses (Fig. 1)Citation . Two human small cell lung carcinoma cell lines (H345, H2081) and two non-small cell lung carcinoma cell lines (H2106, H1838) contained PPAR{gamma} mRNA with variable levels of expression (Fig. 1A)Citation . PPAR{gamma} protein was also detected in all cell lines studied (Fig. 1B)Citation . As expected, phorbol 12-myristate 13-acetate and PGJ2 enhanced the expression of PPAR{gamma} mRNA as shown in H1838 cells (Fig. 1C)Citation .



View larger version (44K):
[in this window]
[in a new window]
  		Fig. 1. Expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in human lung carcinoma cells. Total RNA and cellular protein was isolated from human lung carcinoma cell lines as indicated, then subjected to reverse transcription (RT)-PCR (A) and Western blot (B) analysis using PPAR{gamma} primers and polyclonal antibodies against PPAR{gamma}, respectively. Total RNA was isolated from untreated H1838 cells or cells treated with phorbol 12-myristate 13-acetate (PMA; 100 nM), or PGJ2 (20 µM) for 24 h, then subjected to RT-PCR (C). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls for normalization purposes.

 
Effect of PPAR{gamma} Ligands on Proliferation of Human Lung Carcinoma Cells.
Next, we examined the effect of PPAR{gamma} activation on cell growth in H2106 cells that expressed high levels of PPAR{gamma}. As shown in Fig. 2Citation , the PPAR{gamma} ligands PGJ2 (A) and Cig (B) inhibited the growth of these cells in a dose-dependent manner. Statistical significance was obtained at 10–30 µM of PGJ2 and Cig. Fig. 3Citation shows the time-dependent effects of these agents on H2106 cell growth. PGJ2 and Cig significantly inhibited cell proliferation when compared with untreated cells and cells treated with the PPAR{alpha} agonist WY14643 starting at 48 h of treatment. The PPAR{gamma} ligands showed similar dose- and time-dependent activity on the growth of the other lung carcinoma cell lines (H1838, H2081, and H345; data not shown). The suppressive cell growth effects of relatively low doses of either PGJ2 (10 µM) or Cig (30 µM) for 72 h were reversible when the media was changed to fresh media containing no PPAR{gamma} ligands. However, the cell growth inhibition became irreversible after 72 h treatment or when cells were exposed to higher doses of either PGJ2 (20 µM) or Cig (50 µM; not shown).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Dose-dependent effect of PGJ2 and ciglitazone (Cig) on human lung carcinoma cell growth. H2106 cells were treated with indicated concentrations of either PGJ2 (A) or Cig (B), and cell numbers were determined on day 4. Data are expressed as mean ± SD of three independent experiments. *, P < 0.05 when compared with the untreated cells.

 


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 3. Time-dependent inhibition of human lung carcinoma cell growth. H2106 cells were treated with solvent control, 30 µM ciglitazone (Cig), 20 µM PGJ2 or 5 µM WY14643, and cell numbers were determined at the indicated time period. Data are expressed as mean ± SD of at least three independent experiments. *, indicated P < 0.05 when compared with the untreated cells. Con, control.

 
Effect of PPAR{gamma} Ligands on Induction of Human Lung Carcinoma Cell Apoptosis.
In Fig. 3Citation , we demonstrated that PPAR{gamma} ligands not only inhibited lung carcinoma cell proliferation, but they also reduced the number of cells below the baseline. This suggested the possibility of induction of apoptosis. To test this possibility, we evaluated PPAR{gamma}-treated cells for DNA fragmentation as a marker for cells undergoing apoptosis. H1838 cells showed significant DNA fragmentation after 72 h treatment with Cig when compared with the control (Fig. 4ACitation , Lane 2 versus 1). In H2061 cells, the PPAR{alpha} ligand WY14643 (25 µM), unlike PGJ2, did not induce apoptosis (Fig. 4BCitation , Lane 2 versus 3). H345 and H2081 cells showed a similar apoptotic response when treated with Cig and PGJ2 (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. Effect of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands in induction of human lung carcinoma cell apoptosis. DNA was isolated from the human lung carcinoma cell line H1838 treated with (A) solvent control (Lane 1), 30 µm ciglitazone (Cig; Lane 2) or H2106 cells treated with (B) solvent control (Lane 1), 25 µM WY14643 (Lane 2), 20 µM PGJ2 (Lane 3) in 2% FBS medium for 72 h. The electrophoretic pattern of the DNA ladder was determined by running the DNA on a 1.5% agarose gel in 1x Tris-acetate-EDTA followed by ethidium bromide staining. M indicates DNA marker.

 
Induction of p21 Gene Expression by PPAR{gamma} Ligands.
Having demonstrated that PPAR{gamma} ligands inhibit lung carcinoma cell growth and promote apoptosis, attention focused on the potential mechanisms mediating this effect. Others have demonstrated that PPAR{gamma} ligands induce cell cycle arrest and apoptosis by up-regulation of p21. To determine the relevance of this pathway in our system, we examined the effects of PPAR{gamma} ligands on p21 expression. Both PGJ2 and Cig significantly induced p21 mRNA levels in H1838 cells at 4 h as determined by RT-PCR (Fig. 5ACitation , top) and confirmed by real-time RT-PCR (Fig. 5ACitation , bottom). Cig and PGJ2 increased p21 expression 2.5- and 4-fold, respectively. In accordance with the changes observed in p21 mRNA, Western blot analysis revealed that PGJ2, Cig, troglitazone, and GW1929 (another PPAR{gamma} ligand; Ref. 41 ), induced the expression of p21 protein levels in H2106 cells after an 8-h culture (Fig. 5B)Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Effect of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands on p21 expression. Total RNA and cellular protein was isolated from H1838 cells that were cultured for 4 and 8 h, respectively, in the presence or absence of indicated compounds [30 µM ciglitazone (Cig), 20 µM troglitazone (Trog), PGJ2 and GW1929]. Reverse transcription (RT)-PCR (A, top), real-time RT-PCR (A, bottom) and Western blot analysis (B, top) were performed. B, bottom, was obtained by densitometry. The bar graphs represent the mean ± SD of p21/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or p21/actin of at least three independent experiments. *, significant differences as compared with the vehicle control. Con, control.

 
We also examined the effect of PPAR{gamma} ligands on one of the cell cycle control genes, cyclin D1, and found that the PPAR{gamma} ligands PGJ2 and Cig reduced the expression of cyclin D1 mRNA levels when compared with the control as determined by RT-PCR (Fig. 6ACitation , top) and real-time RT-PCR (Fig. 6ACitation , bottom) at 12 h. The reductions observed were in the range of 60% (for PGJ2) and 65% (for Cig). PPAR{gamma} ligands also significantly reduced cyclin D1 protein levels as compared with the control (Fig. 6B)Citation . Similar results were also found in the other lung carcinoma cell lines studied (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6. Effect of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands on cyclin D1 gene expression in human lung carcinoma cells. Total RNA and cellular protein were isolated from H2106 cells that were treated with PGJ2, troglitazone (Trog; 20 µM), or ciglitazone (Cig; 30 µM) for 12 and 24 h, respectively. Reverse transcription (RT)-PCR (A, top), real-time RT-PCR (A, bottom) and Western blot analysis (B) were performed using cyclin D1 primers and polyclonal antibodies against cyclin D1, respectively. A, bottom, indicates fold changes of cyclin D1 mRNA levels by PPAR{gamma} treatment as compared with control. The bar graphs represent the mean ± SD cyclin D1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or cyclin D1/actin band densities of at least three independent experiments. *, significant differences as compared with the vehicle control. Con, control.

 
PPAR{gamma} Ligands Stimulate p21 Promoter Activity.
We demonstrated that PPAR{gamma} ligands increased p21 gene expression in human lung carcinoma cells. To explore whether this effect reflects increased transactivation of the p21 promoter, transient transfections were performed with human p21 promoter constructs. As shown in Fig. 7Citation , the PPAR{gamma} ligands PGJ2 and Cig were found to significantly activate the 2.3-kb fragment of the p21 promoter, whereas WY14643 had little effect.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 7. Effect of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands on p21 promoter activity. H1838 human lung carcinoma cells were cotransfected with p21 promoter construct ligated to luciferase and 0.2 µg of control Renilla hpRL-SV40 plasmid. After transfection for 24 h, cells were cultured in the presence or absence of indicated compounds [20 µM PGJ2, 30 µM ciglitazone (Cig), or 25 µM WY14643] for another 24 h, after which luciferase activity was determined using dual-report luciferase kit. A representative experiment of at least three performed is shown. *, significant difference from control. Con, control.

 
PPAR{gamma} Ligands Increase the Binding Activities of Sp1 and NF-IL6 (C/EBP).
Next, electrophoretic mobility shift assay was performed to identify potential nuclear transcription factors that could mediate regulation of the p21 gene by the PPAR{gamma} ligands. The human p21 promoter region contains multiple transcription factor binding sites, including Sp1 and binding motifs for NF-IL6 (C/EBP), and proteins that bind to these sites play important roles in cell cycle arrest and apoptosis in carcinoma cells (37 , 40 , 42 , 43) . In agreement with those studies, we found that H2106 cells treated with PPAR{gamma} ligands for 24 h showed an induction in Sp1 (Fig. 8A)Citation and NF-IL6 (C/EBP; Fig. 8BCitation ) binding activities. There was no binding activity when the Sp1 or NF-IL6 (C/EBP) sites were mutagenized. The PPAR{alpha} agonist WY14643 had little effect, suggesting the specificity of the activity of the PPAR{gamma} ligands. In competition assays, we found significant attenuation of Sp1 and NF-IL6 (C/EBP) bands by adding a 100-fold molar excess of unlabeled oligonucleotides [Sp1 and NF-IL6 (C/EBP)]. These results confirm that the PPAR{gamma} ligands (PGJ2 and Cig) can increase the binding levels of Sp1 and NF-IL6 (C/EBP) transcription factor complexes in human lung carcinoma cells.



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 8. Electrophoretic mobility shift assay (EMSA) to determine the Sp1 and CAAT/enhancer binding protein (C/EBP) protein binding activities in p21 promoter affected by peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands. Oligonucleotides containing the Sp1 (A) or C/EBP (B) sites were end labeled with [{gamma}32P]ATP and incubated with nuclear extracts (10 µg) from H2106 cells treated with PPAR{gamma} ligands PGJ2 (20 µM), ciglitazone (Cig; 30 µM), or PPAR{alpha} ligand WY14643 for 24 h. For competition assays, a molar excess (x100) of consensus Sp1 or C/EBP oligonucleotide was added to the binding reaction. Oligonucleotides containing a mutated Sp1 (Cold Sp1) or C/EBP site (Cold C/EBP) that were end labeled with [{gamma}32P]ATP or 100 times molar excess of unlabeled mutated Sp1 oligonucleotide (mut Sp1) were used to confirm the binding specificity. Nuclear extract from HeLa cells was used as positive control for Sp1. Con, control; FP, free probe.

 
p21 Antisense Oligonucleotides Inhibit Both PPAR{gamma} Ligand-Induced p21 Protein Expression and PPAR{gamma} Ligand-Reduced Lung Carcinoma Cell Growth.
An earlier study showed that p21 antisense ODN can reduce p21 protein levels in both animal and human cells (35 , 44) . To determine whether similar effects are observed in lung carcinoma cells, H1838 cells were transfected with p21 phosphorothioate ODN or control random ODN. Afterward, the cells were treated with PPAR{gamma} ligands (PGJ2 and Cig) for another 24 h for Western blot analysis or up to 72 h for cell proliferation assays. Our results showed that the antisense p21 ODN inhibited PPAR{gamma} ligand-induced p21 protein levels by Western blot analysis (Fig. 9)Citation . Random control ODN had no effect (data not shown). Cell growth inhibition assays demonstrated that p21 antisense ODN significantly blocked PGJ2- or Cig-mediated inhibition of cell growth whereas p21 control ODN or p21 antisense ODN alone had little effect (Fig. 10)Citation .



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 9. Antisense p21 oligodeoxynucleotide (ODN) prevents peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand-induced p21 protein levels in human lung carcinoma cells. H1838 cells were treated with solvent control or control and p21 antisense ODNs for 24 h before adding PGJ2 (20 µM) or ciglitazone (Cig; 30 µM) for additional 24 h, then subjected to Western blot analysis. Actin was used as internal control for normalization purposes (A). The bar graphs (B) obtained by densitometry represent the mean ± SD p21/actin of at least three independent experiments. *, significant difference from control. **, significance of combination treatment as compared with PGJ2 or Cig alone. Con, control.

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 10. Antisense p21 oligodeoxynucleotides (ODNs) antagonized peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligand-mediated suppression of carcinoma cell proliferation. H2106 cells were treated with either solvent control or transfected with control ODN or p21 antisense ODN for 24 h before exposing them to 30 µM ciglitazone (Cig) or 20 µM PGJ2 for an additional 48 h as indicated, and cell numbers were determined. Data are expressed as mean ± SD of at least three independent experiments. *, significant difference from control. **, significance of combination treatment as compared with PGJ2 or Cig alone. Con, control.

 
Discussion

PPAR{gamma} ligands have been implicated in the regulation of cell differentiation, apoptosis, and cell cycle control (18) . The expression of PPAR{gamma} and the effect of PPAR{gamma} ligands on cell growth inhibition and apoptosis have been investigated in many cells including lung carcinoma (21 , 23, 24, 25, 26) . Although these studies showed that several PPAR{gamma} ligands can induce cell growth inhibition and apoptosis, little is known about the mechanism(s) mediating these effects on human lung carcinoma cells. Therefore, we investigated whether the cell cycle control genes cyclin D1 and the cyclin-dependent kinase inhibitor p21 are involved in PPAR{gamma}-mediated growth regulation in these cells. We found variable levels of PPAR{gamma} mRNA and protein in four human lung carcinoma cell lines. Treatment with the PPAR{gamma} ligand PGJ2 induced PPAR{gamma} mRNA levels as compared with the control, suggesting that PPAR{gamma} signaling was functional. We next examined the effect of PPAR{gamma} activation on cell growth in these cells. Treatment with PGJ2 and Cig suppressed cell growth in a dose- and time-dependent manner. Of note, we found that the PPAR{gamma} ligands PGJ2, troglitazone, and Cig, but not the PPAR{alpha} agonist WY14643, induced apoptosis of H2106 or H1838 cells. These results expand current knowledge about the proapoptotic effects of PPAR{gamma} ligands on human lung carcinoma cells (13 , 17) .

Expression of cyclin-dependent kinase inhibitors are purported to play important roles in the control of cell cycle in many cell types, including lung carcinoma in vitro and in vivo (45, 46, 47) . Although the exact mechanism of growth inhibition of carcinoma cells by PPAR{gamma} ligands is not known, it has been shown to be associated with alterations in the expression of the cell cyclin-dependent kinase inhibitor p21 (26 , 27) . Consistent with this idea, we showed that PGJ2 and Cig up-regulated the expression of p21 protein and mRNA concomitant with inhibition of cyclin D1 protein and mRNA levels. Inhibition of cyclin D1 gene expression through PPAR{gamma} signaling had also been reported in other cells types as well as lung carcinoma cells (48 , 49) . As such, our results support the hypothesis that suppression of lung carcinoma cell growth through PPAR{gamma} activation may be mediated via reduction of cyclin D1 levels.

p21 has been shown to be regulated at the level of transcription in different cell types. Park et al. (37) showed that mitogen-activated protein kinase signaling increased p21 promoter activity via multiple transcription factors. Li et al. (50) demonstrated that growth inhibition associated with an increase in JunD/AP-1 activity in normal intestinal epithelial cells was at least partially mediated through the activation of the p21 promoter. To investigate whether PPAR{gamma} ligand-mediated up-regulation of p21 reflects transactivation of the promoter, we performed transient transcription experiments using a p21 promoter reporter construct. We found that the PPAR{gamma} ligands PGJ2 and Cig increased p21 promoter activity, whereas the WY14643 had little effect.

Transcriptional regulation of the p21 promoter activity appears to be complex and consists of both positive and negative regulatory elements. Multiple transcription factor binding sites within the p21 promoter have been characterized including regulatory elements for Sp1 and C/EBP family members (37 , 40 , 51) . As such, we evaluated the possibility that Sp1 and C/EBP might play a role in PPAR{gamma} ligand up-regulation of p21 expression. We showed that treatment of H2106 cells with PPAR{gamma} ligands (Cig and PGJ2) enhanced nuclear protein binding activities of Sp1 and C/EBP sites in the p21 promoter. In contrast, negative regulation of C/EBP and Sp1 transcription factor binding activity by PPAR{gamma} has also been described in other cell systems (52 , 53) . Thus, it is apparent that the nature and qualities of PPAR{gamma} effects on Sp1 and C/EBP are highly cell type specific.

Utilization of antisense ODN to p21 in vivo and in vitro has been shown to be a sensitive way to examine its role in inhibition of cell proliferation, angiogenesis, matrix protein production, and induction of apoptosis (35 , 44 , 54) . Our results in p21 antisense experiments showed that this ODN antagonized PPAR{gamma} ligand induction of p21 protein and suppression of lung carcinoma cell growth. These results are consistent with studies by Weiss et al. (55) who showed that antisense ODN for p21 decreased p21 protein levels and affected breast carcinoma cell growth and by Moffatt et al. (56) who found that the p21-mediated antiproliferative effect of 1{alpha}, 25-(OH)2D3 on prostatic carcinoma cells was abolished by stable transfection with a p21 antisense construct.

Taken together, our study indicates that PPAR{gamma} ligand inhibition of human lung carcinoma cell growth and induction of apoptosis are mediated via up-regulation of the cyclin-dependent kinase inhibitor p21 and reduction of cyclin D1 gene expression. Induction of p21 gene expression by PPAR{gamma} ligand appears to be, at least in part, regulated through C/EBP and Sp1 sites in the p21 gene promoter. Although additional studies will be required to determine the clinical implications of our findings, our observations suggest that ligand activation of PPAR{gamma} represents a potential new molecular target for effective therapy against human lung carcinoma. Studies in humans, animals, and in cultured cells support the idea that modulation of PPAR{gamma} activation may have therapeutic benefits in other tumors including colon and pituitary carcinoma (57 , 58) . There was no difference in survival between patients who received adjuvant chemotherapy and those who did not (59) . We speculate that specific PPAR{gamma} ligands used as single agents or as part of combination regimens could serve to further improve outcomes in these patients by inhibiting carcinoma cell growth.

FOOTNOTES

Grant support: American Cancer Society Institutional Research Grant 6–47083 (S. Han), NIH, National Cancer Institute Grants CA85589 (N. Sidell), CA35675 (P. Fisher) and CA97318 (P. Fisher), and a Merit Review Grant from the Department of Veterans Affairs (J. Roman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Shouwei Han, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Whitehead Bioresearch Building, 615 Michael Street, Suite 205-M, Atlanta, GA 30322. Phone: (404) 712-2661; Fax: (404) 712-2151; E-mail: shan2{at}emory.edu

Received 7/ 7/03; revised 11/14/03; accepted 12/ 9/03.

REFERENCES

  1. Fang JY, Lu YY Effects of histone acetylation and DNA methylation on P21 (WAF1) regulation. World J Gastroenterol, 8: 400-5, 2002.[Medline]
  2. Chellappan SS, Giordano A, Fisher PB Role of cyclin dependent kinases and their inhibitors in cellular differentiation and development. Curr Top Microbiol Immunol, 227: 57-103, 1998.[Medline]
  3. Chen J, Saha P, Kornbluth S, Dynlacht BD, Dutta A Cyclin-binding motifs are essential for the function of p21CIP1. Mol Cell Biol, 16: 4673-82, 1996.[Abstract]
  4. Kim JS, Lee S, Lee T, Lee YW, Trepel JB Transcriptional activation of p21 (WAF1/CIP1) by apicidin, a novel histone deacetylase inhibitor. Biochem Biophys Res Commun, 281: 866-71, 2001.[CrossRef][Medline]
  5. Gu Y, Turck CW, Morgan DO Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature (Lond), 366: 707-10, 1993.[CrossRef][Medline]
  6. el-Deiry WS, Tokino T, Velculescu VE, et al WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-25, 1993.[CrossRef][Medline]
  7. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75: 805-16, 1993.[CrossRef][Medline]
  8. Jiang H, Fisher PB Use of a sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the induction of differentiation in human melanoma cells. Mol Cell Differ, 1: 285-99, 1993.
  9. Jiang H, Lin J, Su ZZ, et al The melanoma differentiation associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells. Oncogene, 10: 1855-64, 1995.[Medline]
  10. Jiang H, Lin J, Su ZZ, Collart FR, Huberman E, Fisher PB Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene, 9: 3397-406, 1994.[Medline]
  11. Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, el-Houseini ME Induction of p21 (WAF-1/CIP1) during differentiation. Oncogene, 9: 3389-96, 1994.[Medline]
  12. Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res, 211: 90-8, 1994.[CrossRef][Medline]
  13. Roninson IB Oncogenic functions of tumour suppressor p21 (Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts. Cancer Lett, 179: 1-14, 2002.[CrossRef][Medline]
  14. Bosch M, Gil J, Bachs O, Agell N Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21 (cip1). J Biol Chem, 273: 22145-50, 1998.[Abstract/Free Full Text]
  15. Niklinski J, Niklinska W, Laudanski J, Chyczewska E, Chyczewski L Prognostic molecular markers in non-small cell lung carcinoma. Lung Cancer, 2(Suppl): S53-8, 2001.[CrossRef]
  16. Komiya T, Hosono Y, Hirashima T, et al p21 expression as a predictor for favorable prognosis in squamous cell carcinoma of the lung. Clin Cancer Res, 3: 1831-5, 1997.[Abstract]
  17. Shoji T, Tanaka F, Takata T, et al Clinical significance of p21 expression in non-small-cell lung carcinoma. J Clin Oncol, 20: 3865-71, 2002.[Abstract/Free Full Text]
  18. Rosen ED, Spiegelman BM PPAR. {gamma}: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem, 276: 37731-4, 2001.[Free Full Text]
  19. Nwankwo JO, Robbins ME Peroxisome proliferator-activated receptor-{gamma} expression in human malignant and normal brain, breast and prostate-derived cells. Prostaglandins Leukot Essent Fatty Acids, 64: 241-5, 2001.[CrossRef][Medline]
  20. Bogazzi F, Ultimieri F, Raggi F, et al Peroxisome proliferator activated receptor {gamma} expression is reduced in the colonic mucosa of acromegalic patients. J Clin Endocrinol Metab, 87: 2403-6, 2002.[Abstract/Free Full Text]
  21. Inoue K, Kawahito Y, Tsubouchi Y, et al Expression of peroxisome proliferator-activated receptor (PPAR)-{gamma} in human lung carcinoma. Anticarcinoma Res, 21: 2471-6, 2001.
  22. Capparuccia L, Marzioni D, Giordano A, et al PPAR{gamma} expression in normal human placenta, hydatidiform mole and choriocarcinoma. Mol Hum Reprod, 8: 574-9, 2002.[Abstract/Free Full Text]
  23. Kopelovich L, Fay JR, Glazer RI, Crowell JA Peroxisome proliferator-activated receptor modulators as potential chemopreventive agents. Mol Cancer Ther, 1: 357-63, 2002.[Abstract/Free Full Text]
  24. Sporn MB, Suh N, Mangelsdorf DJ Prospects for prevention and treatment of carcinoma with selective PPAR{gamma} modulators (SPARMs). Trends Mol Med, 7: 395-400, 2001.[CrossRef][Medline]
  25. Chang TH, Szabo E Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor {gamma} in non-small cell lung carcinoma. Cancer Res, 60: 1129-38, 2000.[Abstract/Free Full Text]
  26. Kawa S, Nikaido T, Unno H, et al Growth inhibition and differentiation of pancreatic carcinoma cell lines by PPAR {gamma} ligand troglitazone. Pancreas, 24: 1-7, 2002.[CrossRef][Medline]
  27. Motomura W, Okumura T, Takahashi N, et al Activation of peroxisome proliferator-activated receptor {gamma} by troglitazone inhibits cell growth through the increase of p27KiP1 in human pancreatic carcinoma cells. Cancer Res, 60: 5558-64, 2000.[Abstract/Free Full Text]
  28. Li XS, Rishi AK, Shao ZM, et al Posttranscriptional regulation of p21WAF1/CIP1 expression in human breast carcinoma cells. Cancer Res, 56: 5055-62, 1996.[Abstract/Free Full Text]
  29. Han S, Sidell N RU486-induced growth inhibition of human endometrial cells involves the nuclear factor-{kappa}B signaling pathway. J Clin Endocrinol Metab, 88: 713-9, 2003.[Abstract/Free Full Text]
  30. Miyahara T, Schrum L, Rippe R, et al Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem, 275: 35715-22, 2000.[Abstract/Free Full Text]
  31. Han S, Sidell N Peroxisome-proliferator-activated-receptor {gamma} (PPAR{gamma}) independent induction of CD36 in THP-1 monocytes by retinoic acid. Immunology, 106: 53-9, 2002.[CrossRef][Medline]
  32. Kawakami S, Arai G, Hayashi T, et al PPAR{gamma} ligands suppress proliferation of human urothelial basal cells in vitro. J Cell Physiol, 191: 310-9, 2002.[CrossRef][Medline]
  33. Xiong Y, Connolly T, Futcher B, Beach D Human D-type cyclin. Cell, 65: 691-9, 1991.[CrossRef][Medline]
  34. Han SW, Inoue H, Flowers LC, Sidell N Control of COX-2 gene expression through peroxisome proliferator-activated receptor {gamma} in human cervical carcinoma cells. Clin Cancer Res, 9: 4627-35, 2003.[Abstract/Free Full Text]
  35. Weiss RH, Randour CJ Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21 (Waf1/Cip1). Atherosclerosis, 161: 105-12, 2002.[CrossRef][Medline]
  36. Han SW, Lei ZM, Rao CV Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology, 137: 1791-7, 1996.[Abstract]
  37. Park JS, Qiao L, Gilfor D, et al A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes. Mol Biol Cell, 11: 2915-32, 2000.[Abstract/Free Full Text]
  38. Han S, Wada RK, Sidell N Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor {gamma}. Cancer Res, 61: 3998-4002, 2001.[Abstract/Free Full Text]
  39. Dignam JD, Lebovitz RM, Roeder RG Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res, 11: 1475-89, 1983.[Abstract/Free Full Text]
  40. Koutsodontis G, Moustakas A, Kardassis D The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter. Biochemistry, 41: 12771-84, 2002.[CrossRef][Medline]
  41. Brown KK, Henke BR, Blanchard SG, et al A novel N-aryl Tyrosine activator of peroxisome proliferator-activated receptor-{gamma} reverses the diabetic phenotype of zucker diabetic fatty rat. Diabetes, 48: 1415-24, 1999.[Abstract]
  42. Yan GZ, Ziff EB Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor. J Neurosci, 17: 6122-32, 1997.[Abstract/Free Full Text]
  43. Black AR, Black JD, Azizkhan-Clifford J Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and carcinoma. J Cell Physiol, 188: 143-60, 2001.[CrossRef][Medline]
  44. Tian H, Wittmack EK, Jorgensen TJ p21WAF1/CIP1 antisense therapy radiosensitizes human colon carcinoma by converting growth arrest to apoptosis. Cancer Res, 60: 679-84, 2000.[Abstract/Free Full Text]
  45. Pitterle DM, Jolicoeur EM, Bepler G Hot spots for molecular genetic alterations in lung carcinoma. In Vivo, 12: 643-58, 1998.[Medline]
  46. Milutinovic S, Knox JD, Szyf M DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21 (WAF1/CIP1/sdi1). J Biol Chem, 275: 6353-9, 2001.
  47. Rumi MA, Sato H, Ishihara S, Ortega C, Kadowaki Y, Kinoshita Y Growth inhibition of esophageal squamous carcinoma cells by peroxisome proliferator-activated receptor-{gamma} ligands. J Lab Clin Med, 140: 17-26, 2002.[CrossRef][Medline]
  48. Toyota M, Miyazaki Y, Kitamura S, et al Peroxisome proliferator-activated receptor {gamma} reduces the growth rate of pancreatic carcinoma cells through the reduction of cyclin D1. Life Sci, 70: 1565-75, 2002.[CrossRef][Medline]
  49. Chang TH, Szabo E Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-{gamma} and inhibitors of histone deacetylase in adenocarcinoma of the lung. Clin Cancer Res, 8: 1206-12, 2002.[Abstract/Free Full Text]
  50. Li L, Liu L, Rao JN, et al JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21 after polyamine depletion. Gastroenterology, 123: 764-79, 2002.[CrossRef][Medline]
  51. Chinery R, Brockman JA, Peeler MO, Shyr Y, Beauchamp RD, Coffey RJ Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal carcinoma: a p53-independent induction of p21WAF1/CIP1 via C/EBPß. Nat Med, 3: 1233-41, 1997.[CrossRef][Medline]
  52. Takata Y, Kitami Y, Yang ZH, Nakamura M, Okura T, Hiwada K Vascular inflammation is negatively autoregulated by interaction between CCAAT/enhancer-binding protein-delta and peroxisome proliferator-activated receptor-{gamma}. Circ Res, 91: 427-33, 2002.[Abstract/Free Full Text]
  53. Sugawara A, Uruno A, Kudo M, et al Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-{gamma} via an interaction with Sp1 in vascular smooth muscle cells. J Biol Chem, 277: 9676-83, 2002.[Abstract/Free Full Text]
  54. Zhu GH, Wong BC, Slosberg ED, et al Overexpression of protein kinase C-ß1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells. Gastroenterology, 118: 507-14, 2000.[CrossRef][Medline]
  55. Weiss RH, Marshall D, Howard L, Corbacho AM, Cheung AT, Sawai ET Suppression of breast carcinoma growth and angiogenesis by an antisense oligodeoxynucleotide to p21(Waf1/Cip1). Cancer Lett, 189: 39-48, 2003.[CrossRef][Medline]
  56. Moffatt KA, Johannes WU, Hedlund TE, Miller GJ Growth inhibitory effects of 1{alpha}, 25-dihydroxyvitamin D (3) are mediated by increased levels of p21 in the prostatic carcinoma cell line ALVA-31. Cancer Res, 61: 7122-9, 2001.[Abstract/Free Full Text]
  57. Bull AW The role of peroxisome proliferator-activated receptor {gamma} in colon carcinoma and inflammatory bowel disease. Arch Pathol Lab Med, 127: 1121-3, 2003.[Medline]
  58. Heaney AP, Fernando M, Melmed S PPAR-{gamma} receptor ligands: novel therapy for pituitary adenomas. J Clin Investig, 111: 1381-8, 2003.[CrossRef][Medline]
  59. Johnson BE Adjuvant chemotherapy for non-small-cell lung carcinoma: the end of the beginning. J Natl Cancer Inst (Bethesda), 95: 1422-4, 2003.[Free Full Text]



This article has been cited by other articles:


Home page
 Endocr Relat CancerHome page
D Bonofiglio, H Qi, S Gabriele, S Catalano, S Aquila, M Belmonte, and S Ando
Peroxisome proliferator-activated receptor {gamma} inhibits follicular and anaplastic thyroid carcinoma cells growth by upregulating p21Cip1/WAF1 gene in a Sp1-dependent manner
Endocr. Relat. Cancer, June 1, 2008; 15(2): 545 - 557.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. Xia, S. Nagase, A. G. Montia, S. M. Kalachikov, M. Keniry, T. Su, L. Memeo, H. Hibshoosh, and R. Parsons
BAF180 Is a Critical Regulator of p21 Induction and a Tumor Suppressor Mutated in Breast Cancer
Cancer Res., March 15, 2008; 68(6): 1667 - 1674.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
K. Y. Kim, J. H. Ahn, and H. G. Cheon
Apoptotic Action of Peroxisome Proliferator-Activated Receptor-{gamma} Activation in Human Non Small-Cell Lung Cancer Is Mediated via Proline Oxidase-Induced Reactive Oxygen Species Formation
Mol. Pharmacol., September 1, 2007; 72(3): 674 - 685.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
F.-S. Chou, P.-S. Wang, S. Kulp, and J. J. Pinzone
Effects of Thiazolidinediones on Differentiation, Proliferation, and Apoptosis
Mol. Cancer Res., June 1, 2007; 5(6): 523 - 530.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H.-J. Kim, J.-Y. Kim, Z. Meng, L. H. Wang, F. Liu, T. P. Conrads, T. R. Burke, T. D. Veenstra, and W. L. Farrar
15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits Transcriptional Activity of Estrogen Receptor-{alpha} via Covalent Modification of DNA-Binding Domain
Cancer Res., March 15, 2007; 67(6): 2595 - 2602.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
A. von Knethen, M. Soller, N. Tzieply, A. Weigert, A. M. Johann, C. Jennewein, R. Kohl, and B. Brune
PPAR{gamma}1 attenuates cytosol to membrane translocation of PKC{alpha} to desensitize monocytes/macrophages
J. Cell Biol., February 26, 2007; 176(5): 681 - 694.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J.-S. Annicotte, I. Iankova, S. Miard, V. Fritz, D. Sarruf, A. Abella, M.-L. Berthe, D. Noel, A. Pillon, F. Iborra, et al.
Peroxisome Proliferator-Activated Receptor {gamma} Regulates E-Cadherin Expression and Inhibits Growth and Invasion of Prostate Cancer.
Mol. Cell. Biol., October 1, 2006; 26(20): 7561 - 7574.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
M. A. Peraza, A. D. Burdick, H. E. Marin, F. J. Gonzalez, and J. M. Peters
The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR)
Toxicol. Sci., April 1, 2006; 90(2): 269 - 295.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
T Suzuki, S Hayashi, Y Miki, Y Nakamura, T Moriya, A Sugawara, T Ishida, N Ohuchi, and H Sasano
Peroxisome proliferator-activated receptor {gamma} in human breast carcinoma: a modulator of estrogenic actions.
Endocr. Relat. Cancer, March 1, 2006; 13(1): 233 - 250.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Han and J. Roman
Rosiglitazone suppresses human lung carcinoma cell growth through PPAR{gamma}-dependent and PPAR{gamma}-independent signal pathways.
Mol. Cancer Ther., February 1, 2006; 5(2): 430 - 437.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Han, J. D. Ritzenthaler, B. Wingerd, and J. Roman
Activation of Peroxisome Proliferator-activated Receptor {beta}/{delta} (PPAR{beta}/{delta}) Increases the Expression of Prostaglandin E2 Receptor Subtype EP4: THE ROLES OF PHOSPHATIDYLINOSITOL 3-KINASE AND CCAAT/ENHANCER-BINDING PROTEIN {beta}
J. Biol. Chem., September 30, 2005; 280(39): 33240 - 33249.
[Abstract] [Full Text]